Chalcogenoxanthylium dyes for purging blood pathogens and for photodynamic therapy

ABSTRACT

Provided are chalcogenoxanthylium compounds which can effectively be used as sensitizers in photodynamic therapy, virucides in photodynamic antimicrobial chemotherapy and reversal agents of Pgp function in cancer cells. Further provided is a general method for the preparation of chalcogenoxanthylium compounds.

This application is a continuation of U.S. patent application Ser. No.11/195,393, filed on Aug. 2, 2005, now U.S. Pat. No. 7,906,500 whichclaims priority to U.S. provisional application No. 60/598,043, filed onAug. 2, 2004, the disclosures of which are herein incorporated byreference.

This work was supported by a Grant no. 1-RO1-HL66779-03 from theNational Institutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Blood is the fluid of life carrying oxygen and nutrient, and whennecessary, drugs/pharmaceuticals throughout the body. Following injuryand/or during surgery, blood levels or blood components may need to beincreased in an individual to sustain life. Blood and blood componentstaken from donors. However, in the blood of an infected donor,pathogenic (disease-causing) microbes may be present. Screeningprocesses can remove tainted blood from the blood and blood-componentsupply, but some donors may have been infected only recently and theirblood pathogens may not yet be at a high enough concentration to bedetected by the screening process. These donors increase the risk oftransfusion-transmission of injection.

The risk from pathogen-contaminated blood can be reduced via varioussterilization techniques. Photodynamic therapy (PDT) using light,endogenous oxygen, and a photosensitizer has been successfully utilizedin the treatment of cancer and the same principles can be applied to theremoval of microbes from blood. Irradiation with wavelengths of lightabsorbed by the photosensitizer produces singlet oxygen through theinteraction of the triplet excited state of photosensitizer withground-state oxygen, which is also a triplet. In the treatment of bloodand blood products, photodynamic therapy is known as PhotodynamicAntimicrobial Chemotherapy (PACT). However, compounds which are used forthe treatment of blood products can damage to the blood cells by causinghemolysis, especially after irradiation.

Various dyes have been used with mixed success in PDT and PACT. Astructure at the heart of many dyes, and indeed, many importantchromophores in chemistry and biology, is the xanthylium nucleus. Therhodamine and rosamine dyes (shown in FIG. 1) are representative of thexanthylium class and have been used as laser dyes, fluorescent labels,and fluorescence emission standards where their high fluorescencequantum yields and photostabilities are exploited.[1-5] The rhodaminedyes are also useful fluorescence probes in cell biology studies,showing specific fluorescence staining of mitochondria and other cellorganelles.[6,7] The rhodamines have been found to accumulateselectively in carcinoma cells [8-10] and to be toxic to cancer cellsboth in vitro [10] and in vivo. [11]

One area where the xanthylium dyes have been minimally utilized isphotodynamic therapy (PDT), where their tumor specificity might truly beexploited. [12,13] PDT is a treatment for various cancers that utilizesthe combination of a tumor-specific photosensitizer, light, andmolecular oxygen to induce cellular toxicity, presumably via thegeneration of singlet oxygen. [12] While rhodamine and rosamine dyesexhibit selective uptake in cancer cells, they are poor producers ofexcited-state triplets [14] and, consequently, of singlet oxygen. Thepoor triplet production limits the use of rhodamine and rosamine dyes asphotosensitizers PDT. Furthermore, the rhodamines and rosamines absorblight of wavelengths too short for effective penetration in tissue.[12]

Heavy-atom effects in brominated rhodamine dyes give increased tripletyields and quantum yields for the generation of singlet oxygen [φ(¹O₂)][15-17] relative to unmodified rhodamines. [18] However, wavelengths ofabsorption are little changed relative to their light-atom counterparts.The brominated analogues still target the mitochondria and haveincreased phototoxicity toward cancer cells. [17] Accordingly, there isa need in the areas of PDT and PACT to identify new compounds useful inthese methodologies.

One compound which has been used in photodynamic therapy studies istetramethyl rosamine (TMR-O). While TMR-O has promise in that it hasbeen shown to be transported into the cell cytoplasm, its ultimateability to be effective is in doubt; as with other rosamine dyes, uponirradiation, TMR-O exhibits a high degree of fluorescence at the expenseof singlet oxygen production, the species which damages tumor cells suchthat they die rather than replicate.

On yet another front in the treatment of cancer, multidrug resistance(MDR) of tumor cells, mediated by the plasma membrane proteinP-glycoprotein (Pgp), is a major concern for treatment of primary,metastatic and recurrent cancer. [26-28] Pgp pumps a variety chemicalsand chemotherapeutic agents from tumor cells, resulting in treatmentfailures. [26, 29-31] Tumor cell resistance to a wide assortment ofchemotherapeutic agents can arise from exposure to a single drug makingsubsequent treatments ineffective. [26-27]

The mechanism by which Pgp overexpression is induced during exposure tochemotherapeutics or chemical agents is not fully understood, and mayoccur at the transcriptional level by mechanisms such as geneamplification, gene rearrangement, DNA methylation, promoter mutation orchromatin modification. [32, 33] With any one of these factors,transcription is the key for induction of Pgp and in some cases thiscould be a rapid response to intra/extracellular stimuli. [32]Development of therapeutic interventions at the transcriptional levelcould be advantageous. Currently, the most direct approach to inhibitingPgp function in cancer is at the level of binding and/or the inhibitionof ATP hydrolysis that Pgp is dependent upon for drug efflux from cells.

Many MDR reversal agents, including verapamil, cyclosporin A, andPSC833, have been examined to counteract the mechanisms of drugresistance. [34-36] However, these compounds have significant drawbacks,such as alterations in cell metabolism and their toxicity toward normaltissues. The therapeutic window for these compounds is severelyrestricted because the dose necessary for effective inhibition of Pgp,in many cases, exceeds the minimal toxic concentration in normal tissue.[26, 37,38] Ideally, Pgp modulators would be administered in combinationwith chemotherapeutic agent(s) to increase anti-cancer drug uptake,retention and effectiveness. However, concomitant administration of highdoses of modulators and therapeutic doses of anti-cancer agents haveresulted in unacceptable toxicity requiring chemotherapeutic dosereduction and ineffective treatment. [38]

One source of Pgp inhibitors might be derived from the cationicrhodamine dyes, such as rhodamine 123 (Rh123) and tetramethylrosamine(TMR-O), both structure given directly below. [39-41] These dyes aretransport substrates for Pgp and have been used as fluorescent markersto determine the efficacy of Pgp modulators. [41,42] However, Rh123 andTMR-O do not inhibit Pgp function. [41,43]

While TMR-O is a Pgp substrate, it has not been completely clear whythis is so. Furthermore, variations of TMR-O, such as its sulfur andselenium analogs, are not readily available, as sulfur and seleniumanalogs of xanthylium compounds (chalcogenoxanthylium compounds) aremore difficult to prepare than xanthylium compounds. In particular, theselenium analogs are difficult to prepare with available methods.

SUMMARY OF THE INVENTION

Provided are chalcogenoxanthylium compounds, i.e., selenium and sulfuranalogs of xanthylium compounds. A method for their preparation fromavailable or easily prepared precursors via directed metallationreactions is also provided (FIG. 4). Further provided are selenium andsulfur analogs of TMR-O, as well as other chalcogenoxanthyliumcompounds, which have high efficiency in producing singlet oxygen andcan be used as sensitizing agents in PDT and PACT. Further provided is amethod for their synthesis. Further provided are compounds, TMR-Se andTMR-S having the ability to inhibit Pgp function in cancer cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—Representative examples of rhodamine dyes.

FIG. 2—Effect of chalcogen atom substitution on singlet oxygen yield.

FIG. 3—Examples of chalcogenoxanthylium compounds.

FIG. 4—Synthesis schematic for chalcogenoxanthylium compounds.

FIG. 5—General method for the preparation of chalcogenoxanthyliumcompounds.

FIG. 6. Time course of the uptake of TMR-O (1×10⁻⁵ M) into culturedPgp-expressing CR1R12 cells in the absence (open circles) or thepresence (filled circles) of 7×10⁻⁶ M verapamil. Experimental conditionsare described in detail in the Experimental Section. Each data point,expressed as fmole TMR-O/cell, represents the mean of three separateexperiments. Data for each experiment was calculated from resultsobtained from at least four wells of a 96-well culture plate, bars arethe SEM.

FIG. 7. Comparison of the uptake of 1×10⁻⁵ M TMR-O or TMR-S after 1 hinto cultured, chemo-sensitive AUXB1 and chemo-resistant CR1R12 cellsafter 1-h incubation in the absence (solid columns) or presence (stripedcolumns) of 7×10⁻⁶ M verapamil. Experimental conditions are described indetail in the Experimental Section. Each column, expressed as fmole ofdye/cell, represents the mean of the data obtained from three separateexperiments. Data for each experiment was calculated from resultsobtained from at least four wells of a 96-well culture plate, bars arethe SEM.

FIG. 8. Cell viability of cultured chemo-resistant CR1R12 cells 24 hafter a 2-h incubation with (filled symbol) or without (open symbol)7×10⁻⁶ M verapamil followed by a 2-h incubation with TMR-O (circles),TMR-S (squares) or TMR-Se (triangles) at indicated concentrations(x-axis) and 1-h exposure of the culture plates to light. Experimentaland light exposure conditions are described in detail in theExperimental Section. Each data point represents the mean, expressed aspercent of control cell viability (cells not exposed to verapamil, dyesor light) of at least three separate experiments performed intriplicate, error bars are the SEM. Cells exposed to light alone or dyesalone in the dark using the same conditions showed no significant lossin cell viability.

FIG. 9. Comparison of the effects of TMR-Se and light exposure on thecell viability of cultured chemo-resistant CR1R12 cells (circles) orchemo-sensitive AUXB1 cells (squares) or in the absence (empty symbol)or presence (filled symbol) of 7×10⁻⁶ M verapamil. Experimental andlight exposure conditions are described in detail in the ExperimentalSection. Each data point represents the mean, expressed as percent ofcontrol cell viability (cells not exposed to verapamil, dye or light) ofat least three separate experiments performed in triplicate, error barsare the SEM. Cells exposed to light alone or dye alone in the dark usingthe same conditions showed no significant loss in cell viability.

FIG. 10. Uptake of Calcein AM (CAM) into cultured chemo-resistant CR1R12cells after exposure to the Pgp modulators verapamil (7×10⁻⁶ M or 7×10⁻⁵M) or cyclosporin A (7×10⁻⁶ M) or after cells were exposed to TMR-S orTMR-Se (1×10⁻⁵ M) for 2 h in the dark followed by 5.0 J cm⁻²irradiation. Experimental and light exposure conditions are described indetail in the Experimental Section. Uptake of CAM is measured over a30-min time period and is expressed as the relative rate of uptakemeasured in control cells (cells not exposed to verapamil, CsA, dyes orlight, relative rate of 1.00±0.03). Each column represents the mean ofat least three separate determinations, bars are the SEM.

DETAILED DESCRIPTION OF THE INVENTION

Chalcogenoxanthylium compounds have been found to generally demonstrategreater formation of singlet oxygen in response to photostimulation thantheir oxygen counterparts. Chalcogenoxanthylium compounds which areamino substituted at the 2 and 7 positions, and particularly those whichhave an aryl group as a substituent in the 9-position have shown thepotential for a high degree of virucidal activity. Specifically, TMR-Seand TMR-S have demonstrated high production of singlet oxygen inresponse to photostimulation in in vitro studies. Not only doessubstitution of sulfur or selenium for oxygen in the xanthylium coregive dramatic increases in the production of ¹O₂ in the photostimulatedcompound, but this substitution also gives longer wavelengths ofabsorption and emission and provides highly effective photosensitizersfor PACT. Some compounds of the present invention are illustrated inFIG. 2 and also include compounds 3, 4, RhII-S and 5 in FIG. 3.

Thus, the present invention provides a method for reducing blood orblood product pathogens by incubating blood with a pathogen-purgingamount of one or more compounds of the present invention and exposingthe blood or blood product to radiation, preferably white light, whichexcites the dye molecules to their first electronically excited state.The term “blood products” as used herein refers to whole blood, redblood cells, white blood cells, platelets or other blood fractions andproducts which are derived from or fabricated from blood. The term“blood pathogens” as used herein means viruses and/or bacteria.

For example, TMR-Se, when incubated with red blood cells, was observedto have extremely high extracellular virucidal activity. TMR-S couldalso inactivate extracellular virus, but generally required higherconcentrations and a higher degree of light exposure than TMR-Se.

Additionally some chalcogenoxanthylium compounds, such as TMR-Se andRhII-Se were able to perform as virucides in red blood cell solutionswith little apparent hemolysis following photoactivation of the dye.This lack of hemolysis persists even during storage followingphotoexcitation of the dye. The high extracellular and intracellularinactivation levels and low hemolysis during storage with RhII-Se isunexpected. In a study of over fifty photosensitizers, it was observedthat none could inactivate virus to this degree without significanthemolysis during storage.

Thus, it is believed that chalcogenoxanthylium compounds, especiallythose having an aromatic substituent at the 9-position, can be used aspotent photosensitizers for PDT.

Furthermore, experiments with chalcogenoxanthylium compounds stronglysuggest that TMR-Se and TMR-S are transport substrates for Pgp,similarly to TMR-O, and may act as reversal agents of Pgp function.

TMR-S and TMR-Se and light can increase the intracellular accumulationof the Pgp substrate CAM into chemo-resistant CR1R12 cells. Initialexperiments demonstrate that highly expressing Pgp cells can be alteredby these tetramethylrosamine analogues to take in 2 times more Pgpsubstrate than cells not exposed to the dyes and light. [44] TMR-S andTMR-Se are phototoxic to chemo-resistant CR1R12 cells when a Pgpmodulator is present indicating that uptake ensues and that the dyes areable to reach and cause damage to a critical intracellular sites duringirradiation.

The present invention describes dyes having the following formula:

Wherein R is an alkyl group of 1-8 carbons, aryl, substituted aryl,heteroaryl, substituted heteroaryl group and E is S or Se. The arylgroup may be mono-, di-, or tri-substituted with substituents at theortho, meta, or para positions. Useful substituents include, but are notlimited to, —CO₂Me, —CO₂H, —NMe₂ and other dialkylamino (each alkylgroup independently having 1-8 carbons), —NHEt and other alkylamino (of1-8 carbons), —NH₂, -Me and other alkyl (of 1-8 carbons), —OMe and otheralkoxy (of 1-8 carbons), and various heterocyclic groups such as1,3-oxazole, 1,3-diazole, 4,5-dihydro-1,3-oxazole, and4,5-dihydro-1,3-thiazole.

A⁻ is an anionic group selected from Cl⁻ and other halides, tosylate andother sulfonates, acetate and other carboxylates, hexafluorophosphate,tetrafluoroborate, and the like;

-   wherein R₁′, R₂′, R₁″ and R₂″ of the amino substituents at the 2-    and 7-positions of the chalcogenoxanthylium core may be the same or    different and may be selected from hydrogen and branched or straight    alkyl groups having eight or fewer carbon atoms. R1′ and R2′ and/or    R1″ and R2″ may be connected such that, together with the attached    nitrogen, a 3, 4, 5, 6 or 7 member ring structure, optionally    bearing alkyl substituents, is formed. For example, the ring    structure may be a substituted piperidine.

The compounds may bear W, X, Y and Z substituents which are,independently, hydrogen or C₁ through C₈ alkyl join the nitrogensubstituents such that a single or double ring structure is formed oneither or both end rings of the molecule. The rings which comprise thenitrogen substituents and substituents W, X, Y and Z may be 5, 6 or 7member rings, for example. The structure below illustrates a compoundhaving a two ring structure on one xanthylium end ring of the compound.

The substitution on the aryl ring may also be a ring structure such asin compounds having the following formula:

The compounds of the present invention also include spirolactonecompounds having the following structure, in which E is preferably Se:

Such spirolactone compounds generally exist in equilibrium with theirring-opened form, in relative proportions which depend upon the pH oftheir environment, with the proportion of spirolactone increasing withpH.

The present invention also provides a method for the synthesis of sulfurand selenium analogues of xanthylium compounds, i.e., compounds in whichthe oxygen in the xanthylium backbone is replaced by sulfur or selenium.Some compounds of the present invention are illustrated below, as wellas in FIG. 2. In particular the sulfur and selenium analogs oftetramethylrosamine (TMR-S and TMR-Se, respectively) can be effectivelysynthesized according to the method of the present invention.

In particular, the compounds of the present invention e.g.,chalcogenoxanthylium compounds, including the thio and seleno analoguesof tetramethylrosamine, are prepared by thedirected-metallation/cyclization of the corresponding N,N-diethyl2-(3-dimethylaminophenylchalcogeno)-4-dimethylaminobenzamide to the2,7-bis-(N,N-dimethylamino)-9H-chalcogenoxanthen-9-one followed by theaddition of phenylmagnesium bromide, dehydration and ion exchange to thechloride salt. The thio and seleno tetramethylrosamines had longerwavelengths of absorption and higher quantum yields for the generationof singlet oxygen than tetramethylrosamine. Both the thio and selenoanalogues of tetramethylrosamine can be used for PACT.

A general method for the preparation of the compounds disclosed above isoutlined below, and comprises the following steps:

1) Providing a compound (1) of the following structure:

I)

-   -   i) Y and Z are hydrogen;    -   ii) R₁′ and R₂′, are independently hydrogen or C₁ through C₈        branched or unbranched alkyl groups and, optionally, R₁′ and R₂′        are alkyl groups connected such that they comprise a three,        four, five, six or seven-membered ring:

-   -    which, if desired, can bear substituents such as, for example,        alkyl or aryl groups;        or

II)

-   -   i) Y and Z are independently hydrogen or C₁ through C₈ alkyl and        R₁′ and R₂′ are independently hydrogen or C₁ through C₈ alkyl;    -   wherein R₁′ and Y are connected such that they comprise a five,        six or seven-membered ring:

-   -    and/or R₂′ and Z are connected such that they comprise a five-,        six- or seven-membered ring:

Compounds of the above structure (1) can be obtained commercially orprepared from appropriately substituted carboxylic acid derivatives byfirst forming the corresponding acid chloride with oxallyl chloride orthionyl chloride and then treating with diethylamine. It is notessential that the substituents on the amide nitrogen be ethyl groups,and other alkyl groups, such as branched and unbranched alkyl groups,such as those comprising eight or fewer carbons can be used instead ofethyl groups.

2) Forming a reaction mixture comprising compound (1),N,N,N,N-tetramethylethylenediamine (TMEDA), sec- or tent-butyl lithiumand a solvent whereby said compound (1) is lithiated at the 2 position.The reaction mixture is generally formed by first combining compound (1)with TMEDA in a solvent, followed by the addition of the butyl lithiumcomponent. The butyl lithium component is preferably added in acontrolled manner, such as by dropwise addition to the solutioncontaining the other reactants. A range of solvents can be used,including for example such common solvents as Tetrahydrofuran (THF),ether, and dimethoxyethane. THF is preferred.

3) Adding a compound (2) of the following structure:

to the reaction mixture such that a compound (3), of the followingstructure is formed:

wherein E is S or Se, and

I)

-   -   i) W and X are hydrogen;    -   ii) R₁″ and R₂″ are independently hydrogen, C₁ through C₈        branched or unbranched alkyl and, optionally, R₁″ and R₂″ are        alkyl groups connected such that they comprise a three, four,        five, six or seven-membered ring:

-   -    which, if desired, can bear substituents such as alkyl or aryl        groups;        or

II)

-   -   i) W and X are independently hydrogen or C₁ through C₈ alkyl;    -   ii) R₁″ and/or R₂″ are independently hydrogen or C₁ through C₈        alkyl;    -    and wherein R₁″ and W are connected such that they comprise a        five, six or seven-membered ring:

-   -    and/or wherein R₂″ and X are connected such that they comprise        a five-, six- or seven-membered ring:

As with R′₁ and R′₂, R″₁ and R″₂ are independently hydrogen or straightor branched alkyl groups, preferably having 8 or fewer carbon atoms.

Compound (2), referred to herein as a “dichalcogenide” compound, can beprepared by the use of a 3-Bromoaniline (optionally N-substituted withone or two alkyl groups, branched or straight, each having 8 or fewercarbons), magnesium, and elemental sulfur or selenium, such as sulfurpowder, selenium metal, whole or crushed selenium shot, seleniumshavings, etc., to form a 3-chalcogenolated aniline compound. Thebromoaniline compound and magnesium are first reacted to form a Grignardreagent. The Grignard reagent is then reacted with the elemental sulfuror selenium. Preferably, the 3-bromoaniline compound, the magnesium anda solvent are combined and refluxed for a time, preferably in the rangeof 0.5 h to 4 h, after which the mixture is cooled, preferably to roomtemperature. Elemental sulfur or selenium is then added to the solutioncontaining the Grignard reagent. The mixture is again refluxed for atime in the range of from 0.5 h to 4 h, and cooled, preferably to aboutroom temperature or below, such as by allowing it to stand for a time.It is then diluted with water and cooled further, both of which can beaccomplished simultaneously by pouring the mixture over ice. An acidsuch as HCl is added to the mixture to give a 3-chalcogenolatedcompound, after which the 3-chalcogenolated compound is oxidized to thedichalcogenide compound. The oxidation is preferably accomplished bycontacting the dichalcogenolated compound with a mild oxidant such asair or an alkylated or arylated selenoxide or telluroxide, such asdihexyl telluroxide. It is preferred to maintain a temperature in therange of 0 to −100° C. during the addition of the butyl lithium and thedichalcogenide.

The dichalcogenide compound is added to the reaction mixture, preferablyin a controlled manner, such as by dropwise addition while dissolved ina suitable solvent, such as THF. It is preferred that the dichalcogenidebe added after the butyl lithium. It is also preferred to reflux for atime greater than 0.5 h after the addition of the butyl lithium compoundand before dichalcogenide addition.

4) Contacting said compound (3) with lithium diisopropylamide (LDA) toform a compound (4) of the following structure:

Compound (3) is contacted with LDA, preferably by dissolving compound(3) in a solvent to form a first solution, and adding LDA, preferablysolvated as a second solution, and stirring the combined for a time inthe range of 0.1 to 2 hours, followed by quenching with a quencher,preferably a saturated ammonium chloride solution.

5) converting compound (4) to a compound (5)of the following structure:

wherein R is an alkyl group of 1-8 carbons, aryl, substituted aryl,heteroaryl, or substituted heteroaryl group and E is S or Se. The arylgroup may be mono-, di-, or tri-substituted with substituents at theortho, meta, or para positions. Useful substituents include, but are notlimited to, —CO₂Me, —CO₂H, —NMe₂ and other dialkylamino (each alkylgroup independently having 8 or fewer carbons), —NHEt and otheralkylamino (of 1-8 carbons), —NH₂, -Me and other alkyl (of 1-8 carbons),—OMe and other alkoxy (of 1-8 carbons), and various heterocyclic groupssuch as 1,3-oxazole, 1,3-diazole, 4,5-dihydro-1,3-oxazole, and4,5-dihydro-1,3-thiazole.

A⁻ is an anionic group such as Cl⁻ or other halides, tosylate or othersulfonates, acetate or other carboxylates, hexafluorophosphate,tetrafluoroborate, and the like.

It may be convenient to sequentially perform two or more of thepreceding steps in the “same pot.” However, it is preferred thatresulting compounds (1), (2), (3) and (4) be purified to some degreebefore their use in a subsequent step, which may be accomplishd bymethods known in the art.

The preferred method of conversion of a compound (4) to compound (5)depends upon the R group desired in the end product. However, theconversion can generally be accomplished by methods including the use ofGrignard-type reagents or organolithium reagents. For example alkylmagnesium bromides and/or alkyl lithiums and phenyl magnesium bromidesand/or aryl lithiums can be used to add an alkyl or a phenyl group,respectively, at the ketone carbon chain of compound (4). Substitutedaryl groups or heteroaryl groups can also be added by the use ofGrignard-type reagents in appropriate solvents. As is well known in theart, the treatment of a ketone compound with either a Grignard reagentor an organolithium reagent, followed by acidification of the solutionresults in the formation of an alcohol compound. In order to prepare achalcogenoxanthylium compound of Formula I, it is necessary to convertthe alcohol to a chalcogenoxanthylium salt. Counter anions includehalides, such as, for example, Cl⁻; sulfonates such as, for example,tosylate; carboxylates, such as, for example acetate;hexafluorophosphate; tetrafluoroborate; and the like. Salt formation canbe accomplished by the addition of the acid of the desired counteranion. For example, the addition of hydrochloric acid orhexafluorophosphoric acid to the solution following Grignard treatmentwill give the chloride and hexafluorophosphate salts, respectively. Theacid is preferably added to the solution in a controlled manner, suchas, for example, dropwise addition. The chalcogenoxanthylium chloride orhexafluorophosphate salt can be precipitated from the solution bycooling the solution to a temperature in the range of −50° C. to 0° C.

Further changing of the anion identity, such as conversion ofhexafluorophosphate salt to a chloride salt can be accomplished bymethods known in the art, such as, for example, ion exchange resins andthe like. The foregoing method is illustrated in FIG. 4.

The following examples describe the synthesis of the compounds of thepresent invention.

General experimental: Solvents and reagents were used as received fromSigma-Aldrich Chemical Co (St. Louis, Mo.) unless otherwise noted.Tetramethylrosamine (TMR-O) was purchased from Molecular Probes, Inc.Cell culture media and antibiotics were obtained from Grand IslandBiological (Grand Island, N.Y.). Fetal bovine serum (FBS) was purchasedfrom Atlanta Biologicals (Atlanta, Ga.). Concentration in vacuo wasperformed on a Büchi rotary evaporator. Chalcogenoxanthones 1 and 2 wereprepared as shown in Scheme 1 in FIG. 4 by a method described herein.NMR spectra were recorded on a Varian Inova 500 instrument with residualsolvent signal as internal standard. UV-visible-near-IR spectra wererecorded on a Perkin-Elmer Lambda 12 spectrophotometer equipped with acirculating constant-temperature bath for the sample chambers. Elementalanalyses were conducted by Atlantic Microlabs, Inc.

EXAMPLE 1

Preparation of Di-3-(N,N-dimethylamino)phenyl Disulfide (4):3-Bromo-N,N-dimethylaniline (1.0 g, 5.0 mmol) was added to a stirredmixture of Mg turnings (0.243 g, 10.0 mmol) in 10 mL of anhydrous THF.The resulting mixture was heated reflux for 2 h and then cooled toambient temperature. Sulfur powder (0.39 g, 5.0 mmol) was added and theresulting mixture was heated at reflux for 2 h. The reaction mixture wascooled to ambient temperature and then poured over 6 g of ice. Ten mL of5% HCl was added and the resulting mixture was filtered through a pad ofCelite. The crude thiol was oxidized via the addition of 0.074 g (0.24mmol) of dihexyltelluride and 10 mL of 30% H₂O₂. [23] After stirring for1 h, the reaction mixture was poured into 50 mL of water and the crudeproduct was extracted with ether (3×25 mL). The combined organicextracts were washed with brine, dried over MgSO₄, and concentrated. Thecrude oil was purified via chromatography on SiO₂ eluted with 10%cyclohexane/CH₂Cl₂ to give 0.79 g (52%) of the disulfide 4 as a whitepowder, mp 91-92° C.: ¹H NMR (CD₂Cl₂) δ 7.81 (d, 2 H, J=6.7 Hz), 7.21(d×d, 1 H, J=2.0, 6.7 Hz), 3.35 (s, 12 H); HRMS (EI), m/z 304.1065(Calcd for C₁₆H₂₀N₂S₂: 304.1068). Anal. Calcd for C₁₆H₂₀N₂S₂: C, 63.12;H, 6.62; N, 9.20. Found: C, 63.08; H, 6.62; N, 9.23.

EXAMPLE 2

Preparation of N,N-Diethyl4-N,N-Dimethylamino-2-[3-(N,N-dimethylamino)-phenylthio]benzamide (6):.sec-Butyllithium (1.3 M in cyclohexane, 2.7 mL, 3.6 mmol) was addeddropwise to a stirred solution of N,N-diethyl 4-N,N-dimethylaminobenzamide (0.79 g, 3.6 mmol) and N,N,N,N-tetramethylethylenediamine(TMEDA, 0.42 g, 3.6 mmol) in 25 mL of anhydrous THF at −78° C. After 1 hat −78° C., disulfide 4 (1.43 g, 3.6 mmol) in 5 mL of THF was addeddropwise. After 0.5 h at −78° C., the reaction mixture was warmed toambient temperature. Ten mL of saturated NH₄Cl was added and theproducts were extracted with CH₂Cl₂ (3×30 mL). The combined organicextracts were washed with brine, dried over MgSO₄ and concentrated. Thecrude product was purified via chromatography on SiO₂ eluted with 20%ether/CH₂Cl₂ to give 0.25 g (51%) of 6 as a pale yellow oil: ¹H NMR(CD₂Cl₂) δ 7.17 (t, 1 H, J=8 Hz), 7.08 (d, 1 H, J=8.5 Hz), 6.81 (t, 2 H,J=2 Hz), 6.69 (d, 1 H, J=8 Hz), 6.63 (d×d, 1 H, J=2, 8.5 Hz), 6.60 (d, 1H, J=2 Hz), 6.58 (d×d, 1 H, J=2, 8.5 Hz); 3.50 (exchange broadened s,4H), 3.18 (s, 6H), 2.86 (exchange broadened s, 6H); ¹³C NMR (CD₂Cl₂) δ169.5, 151.4, 151.0, 135.7, 133.8, 129.7, 127.5, 127.2, 119.7, 115.6,115.0, 111.6, 111.0, 43.2 (br), 40.5, 40.2, 14.2 (br); IR (film, NaCl)1621, 1594 cm⁻¹; HRMS (ES) m/z 372.2109 (Calcd for C₂₁H₃₀N₃OS+H:372.2104).

EXAMPLE 3

Preparation of 2,7-Bis-N,N-(dimethylamino)thioxanthen-9-one (2): To asolution of 6 (0.52 g, 1.5 mmol) in 10 mL of THF at 25° C. was added LDA(1.0 M in hexanes, 3.6 mL, 3.6 mmol). The resulting mixture was stirredat ambient temperature for 15 h and was quenched by the addition of 20mL of saturated NH₄Cl. The products were extracted with CH₂Cl₂ (3×10 mL)and the combined organic extracts were washed with brine, dried overNa2SO₄, and concentrated. The products were purified via chromatographyon SiO₂ eluted with 10% ether/CH₂Cl₂ to give 0.37 g (70%) of recovered 6and 0.52 g (13%) of 2 as a yellow powder, mp 260-261° C.: ¹H NMR [500MHz, CD₂Cl₂] δ 8.33 (d, 2 H, J=9.2 Hz), 6.80 (d×d, 2 H, J=2.1, 9.2 Hz),6.77 (d, 2 H, J=2.1 Hz), 3.07 (s, 12 H); ¹³C NMR (CD₂Cl₂) 177.2; 151.7,138.6, 130.2, 118.5, 110.9, 104.8, 39.6; IR (KBr) 1592 cm⁻¹; HRMS (ES)m/z 299.1217 (Calcd for C₁₇H₁₈ON₂S+H: 299.1213).

EXAMPLE 4

Preparation of 2,7-Bis-N,N-dimethylamino-9-phenylselenoxanthyliumChloride (TMR-Se): A solution of2,7-bis-N,N-dimethylamino-9H-selenoxanthen-9-one (1, 0.070 g, 0.20 mmol)in THF (3 mL) was added dropwise to a solution of phenylmagnesiumbromide (1.0 mL of a 1.0 M solution in THF, 1.0 mmol) in THF (2 mL)heated to reflux. After addition was complete, the resulting solutionwas heated at reflux for an additional 0.5. The reaction mixture wasthen cooled to ambient temperature and poured into acetic acid (3.0 mL).Hexafluorophosphoric acid (60% weight solution in water) was addeddropwise until the initial deep blue solution turned reddish yellow.Water (50 mL) was added and the resulting mixture was cooled at −10° C.precipitating the selenoxanthylium hexafluorophosphate salt. The dye wascollected by filtration and the solid was washed with water (2×5 mL) anddiethyl ether (2×5 mL). The crude product was recrystallized bydissolving the solid in 2 mL of hot CH₃CN, cooling to ambienttemperature, slowly adding 2 mL of ether, and chilling. Therecrystallized product was collected by filtration, washed with ether(2×5 mL), and dried to give 0.109 g (98%) of the hexafluorophosphatesalt as a dark green solid: mp>260° C.; ¹H NMR [500 MHz, CD₂Cl₂] δ 7.62(m, 3 H) 7.44 (d, 2 H, J=9.8 Hz), 7.30 (m, 2 H), 7.27 (d, 2 H, J=2.5Hz), 6.83 (d×d, 2 H, J=2.5, 9.8 Hz), 3.25 (s, 12 H); ¹³C NMR [500 MHz,CD₂Cl₂] δ 161.3, 152.6, 146.1, 138.1, 136.9, 128.9, 128.8, 128.3, 119.6,114.4, 109.2, 40.4; λ_(max) (CH₂Cl₂) 582 nm (ε=6.9×10⁴ M⁻¹ cm⁻¹); HRMS(ES) m/z 407.1038 (Calcd for C₂₃H₂₃N₂ ⁸⁰Se: 407.1026). Anal. Calcd forC₂₃H₂₃F₆N₂OPSe: C, 50.10; H, 4.20; N, 5.08. Found: C, 50.20; H, 4.32; N,4.96.

The hexafluorophosphate salt (0.109 g, 0.068 mmol) was dissolved in 20mL of acetonitrile and 0.500 g of Amberlite IRA-400 Chloride ionexchange resin was added. The resulting mixture was stirred 0.5 h, theexchange resin was removed via filtration, and the process was repeatedwith two additional 0.500-g aliquots of the ion exchange resin.Following the final ion exchange, the filtrate was concentrated and thesolid residue was recrystallized from acetonitrile and a small amount ofdiethyl ether to give 0.081 g (75%) of2,7-bis-N,N-dimethylamino-9-phenylselenoxanthylium chloride (TMR-Se) asa dark purple solid: mp>260° C.; ¹H NMR [500 MHz, CD₂Cl₂] δ 7.63 (m, 3H), 7.44 (d, 2 H, J=6.1), 7.30 (d×d, 2 H, J=1.8, 7.3 Hz), 7.28 (d×d, 2H, J=2.1, 7.3 Hz), 6.83 (d×d, 2 H, J=2.1, 9.1 Hz), 3.25 (s, 12 H);λ_(max) (H₂O) 580 nm (ε=5.9×10⁴ M⁻¹ cm⁻¹); HRMS (ES) m/z 407.1038 (Calcdfor C₂₃H₂₃N₂ ⁸⁰Se: 407.1026). Anal. Calcd for C₂₃H₂₃ClN₂OSe: C, 62.52;H, 5.25; N, 6.34. Found: C, 62.46; H, 5.08; N, 6.28.

EXAMPLE 5

Preparation of 2,7-Bis-N,N-dimethylamino-9-phenylthioxanthylium Chloride(TMR-S): A solution of 2,7-bis-N,N-dimethylamino-9H-thioxanthen-9-one(2, 0.050 g, 0.17 mmol) in THF (3 mL) was treated with phenylmagnesiumbromide (1.0 mL of a 1.0 M solution in THF, 1.0 mmol) in THF (2 mL) asdescribed for TMR-Se. Workup and recrystallization gave 0.078 g (71%) of2,7-bis-N,N-dimethylamino-9-phenylthioxanthylium hexafluorophosphate:mp>260° C.; ¹H NMR [500 MHz, CD₂Cl₂] δ 7.68 (m, 3 H), 7.47 (d, 2 H,J=9.8 Hz), 7.37 (m, 2 H), 7.14 (d, 2 H, J=2.4 Hz), 6.95 (d×d, 2 H,J=2.4, 9.8 Hz), 3.31 (s, 12 H); ¹³C NMR [500 MHz, CD₂Cl₂] δ 153.5,144.4, 136.5, 135.4, 129.5, 129.2, 128.7, 119.2, 115.1, 114.2, 105.4,40.5; λ_(max) (CH₂Cl₂) 571 nm (ε=5.0×10⁴ M⁻¹ cm⁻¹); HRMS (ESI) m/z359.1580 (Calcd for C₂₃H₂₃N₂S: 359.1582). Anal. Calcd for C₂₃H₂₃F₆N₂OPS:C, 54.76; H, 4.60; N, 5.55. Found: C, 54.75; H, 4.75; N, 5.24.

The hexafluorophosphate salt (0.045 g, 0.068 mmol) was treated withAmberlite IRA-400 Chloride ion exchange resin as described for thepreparation of TMR-Se to give 0.026 g (78%) of2,7-bis-N,N-dimethylamino-9-phenylthioxanthylium chloride (TMR-S) as adark green, crystalline solid: mp>260° C.; ¹H NMR [500 MHz, CD₂Cl₂] δ7.67 (m, 3 H), 7.46 (d, 2 H, J=9.1 Hz), 7.36 (m, 2 H), 7.14 (d, 2 H,J=2.5 Hz), 6.95 (d×d, 2 H, J=2.5, 9.1 Hz), 3.31 (s,12 H); λ_(max) (H₂O)570 nm (ε=4.0×10⁴ M⁻¹ cm⁻¹); HRMS (ESI), m/z 359.1579 (Calcd forC₂₃H₂₃N₂S: 359.1582). Anal. Calcd for C₂₃H₂₃ClN₂OS: C, 69.94; H, 5.87;N, 7.07. Found: C, 70.03; H, 5.75; N, 7.05.

EXAMPLE 6

Synthesis of 2,7-bis-N,N-dimethylamino-9H-thioxanthen-4-one (2): Thesynthesis of 2,7-bis-N,N-dimethylamino-9H-selenoxanthen-4-one (1)(numbers in this example refer to FIG. 4) from acyclic precursors viadirected metallation reactions is shown in Scheme 1 (FIG. 4).¹⁹ The sameapproach was used for the synthesis of2,7-bis-N,N-dimethylamino-9H-thioxanthen-4-one (2). Lithiation ofN,N-diethyl 4-dimethylaminobenzamide with tert-BuLi in the presence ofN,N,N′,N′-tetramethylethylenediamine (TMEDA) followed by the addition ofdi-(3-dimethylamino)phenyl diselenide (3) or disulfide (4) gave2-arylselenobenzamide 5 or 2-arylthiobenzamide 6 in 57 and 51% isolatedyields, respectively. Cyclization of 5 and 6 with two equivalents oflithium diisopropylamide (LDA) gave selenoxanthenone 1 in 23% yield andthioxanthenone 2 in 13% yield, respectively, with 70% recovered startingmaterial in each case. The starting material could be recycled to givemore 1 and 2. Use of larger excesses of LDA gave up to 30% yields of 1and 2, but no starting material was recovered under these conditions.

The addition of phenylmagnesium bromide to chalcogenoxanthen-9-ones 1and 2 followed by dehydration with HPF₆ gave2,7-bis-N,N-dimethylamino-9-phenylselenoxanthylium and2,7-bis-N,N-dimethylamino-9-phenylthioxanthylium hexafluorophosphates in98 and 71% isolated yields, respectively. Anion exchange with AmberliteIRA-400 chloride exchange resin gave the corresponding chloride saltsTMR-Se, and TMR-S in 78 and 75% isolated yields, respectively.

The following example demonstrates that the use of sulfur and seleniumanalogs of anthylium-type compounds results in increased production ofsinglet oxygen with respect to TMR-O. Increased singlet oxygenconcentration corresponds to greater efficacy of the xanthylium compoundas a PDT and PACT agent. In addition, the cyclic voltammetry resultsshow that TMR-S and TMR-Se are more difficult to reduce than AA1, acompound which has been successfully used in PDT. This result indicatesthat reduction (inactivation) of TMR-S and TMR-Se in the mitochondria isunlikely to occur to such a degree as to compromise the usefulness ofTMR-S and TMR-Se as PDT and PACT agents.

EXAMPLE 7

Experimental

Quantum Yield Determinations for the Generation of Singlet Oxygen: Thequantum yields for singlet-oxygen generation with chalcogenoxanthyliumdyes TMR-O, TMR-S were measured by direct methods in MeOH.²⁴ A SPEX 270Mspectrometer (Jobin Yvon) equipped with InGaAs photodetector(Electro-Optical Systems Inc., USA) was used for recordingsinglet-oxygen emission spectra. A diode pumped solid-state laser(Millenia X, Spectra-Physics) at 532 nm was the excitation source. Thesample solution in a quartz cuvette was placed directly in front of theentrance slit of the spectrometer and the emission signal was collectedat 90-degrees relative to the exciting laser beam. An additionallongpass filter (850LP) was used to attenuate the excitation laser andthe fluorescence from the photosensitizer.

Fluorescence Quantum Yields and Radiative Lifetimes: Fluorescencequantum yields (φ_(F)) and radiative lifetimes (τ_(rad)) were determinedusing techniques and equipment described in Baker, G. A.; Bright, F. V.;Detty, M. R.; Pandey, S.; Stilts, C. E.; Yao, H. R. J. PorphyrinsPhthalocyanines 2000, 4, 669. [25] The results are shown in Table I.

Measurement of Reduction Potential: A BAS 100 potentiostat/galvanostatand programmer were used for the electrochemical measurements. Theworking electrode for cyclic voltammetry was a platinum disk electrode(1-mm diameter) obtained from Princeton Applied Research. The auxiliaryand reference electrodes were silver wires. The reference for cyclicvoltammetry was the ferrocene/ferrrocinium couple at +0.40 V at a scanrate of 0.1 V s⁻¹. All samples were run in J. T. Baker HPLC-gradedichloromethane that had been stored over 3A molecular sieves andfreshly distilled prior to use. Tetrabutylammonium fluoroborate(Sigma-Aldrich) was recrystallized from EtOAc—ether and then driedovernight at 80° C. under vacuum before it was used as supportingelectrolyte at 0.2 M. Nitrogen was used for sample deaeration.

Photophysical Properties: Photophysical properties of TMR-S and TMR-Seare compared to those of commercially available tetramethylrosamine(TMR-O, Molecular Probes, Inc.) in Table 1. In MeOH, a 20-nmbathochromic shift for TMR-S and a 30-nm bathochromic shift for TMR-Sein the wavelength of the absorption maximum, λ_(max), is observed as thechalcogen atom becomes larger and the oscillator strength of the banddecreases as indicated by a decrease in the molar extinctioncoefficient, ε.

TABLE 1 Absorption (λ_(max)) and Fluorescence (λ_(F)) Maxima in MeOH,Quantum Yields for Fluorescence (φ_(F)) and the Generation of SingletOxygen [φ(¹O₂)], Reduction Potentials (E°), and n-Octanol/WaterPartition Coefficients (log P) for TMR-O, TMR-S, and TMR-Se. λ_(max), nmCompd (log ∈) λ_(F), nm^(a) φ_(F) ± sd^(b) φ(¹O₂) ± sd^(c) E°, V^(d) logP^(e) TMR-O 552 (4.92) 575 0.84 ± 0.01 0.08 ± 0.01 −0.94 1.5 TMR-S 571(4.70) 599 0.44 ± 0.01 0.21 ± 0.02 −0.79 1.2 TMR-Se 582 (4.84) 608 0.009± 0.001 0.87 ± 0.01 −0.77 1.1 ^(a)Excitation at 532 nm. ^(b)Quantumefficiencies using rhodamine 6G as a standard. ^(c)Direct detection ofsinglet-oxygen luminescence using rose bengal as a standard. ^(d)InCH₂Cl₂ with 0.2N Bu₄NBF₄ as supporting electrolyte. V vs. theferrocene/ferrocinium couple (E° = +0.40 V). ^(e)pH-6 phosphate bufferas the aqueous phase.

Results

A 25- to 28-nm Stokes shift is observed in the fluorescence emissionmaximum, λ_(F), which is typical of other cationic dyes. [20] Theradiative lifetime, τ_(rad), decreases from 2.1±0.1 ns in TMR-O to1.5±0.1 ns for TMR-S to approximately 50 ps for TMR-Se, which isconsistent with heavy-atom effects in other cationic dyes. [20]

The rigid nature of the xanthylium core minimizes internal conversionback to the ground state, which leads to dramatic changes in φ_(F) andφ(¹O₂) as the chalcogen atom becomes larger. Fluorescence in TMR-O ishighly efficient with φ_(Φ) of 0.84. Intersystem crossing to thetriplet, which is necessary for the production of singlet oxygen, isrelatively slow with φ(¹O₂) of 0.08 for TMR-O. As the chalcogen atombecomes larger, the spin-orbit (heavy-atom) effects promote tripletformation relative to fluorescence and φ(¹O₂) decreases to 0.44 and0.009 for TMR-S and TMR-Se, respectively, while values of φ(¹O₂)increase to 0.21 and 0.87 for TMR-S and TMR-Se, respectively.

Chemical Properties: Mitochondrial reduction of a cationicphotosensitizer can bleach the photosensitizer, thus limiting it'suseful lifetime. Furthermore, the cationic photosensitizer's role as anelectron acceptor might impact the redox cascade in mitochondrialrespiration. The reduction potentials of TMR-O, TMR-S, and TMR-Se weredetermined by cyclic voltammetry and values of E° for the cation/neutralradical couple [vs. the ferrocene/ferrocinium couple (Fc/Fc⁺) at +0.40V] are compiled in Table 1. An anodic (positive) shift in E° is observedas the chalcogen atom becomes heavier. The reduction of thetetramethylrosamines is more cathodic (more difficult to reduce) thanthe thiopyrylium dye AA1 [E° of −0.71 V (vs. Fc/Fc⁺)], which showsantitumor activity by targeting the mitochondria of carcinoma cells [21]and inhibiting mitochondrial ATPase activity. [21]

EXAMPLE 8

This example demonstrates that the compounds of the present inventioncan be used for PACT.

Preparation of Oxygenated Red Blood Cells: Packed RBCs were preparedfrom units of whole blood (500±50 mL) collected in 70 mL CDP intriple-pack container systems (PL146 primary container, BaxterHealthcare, Deerfield, Ill.) by the Research Blood Department, HollandLaboratory for the Biomedical Sciences. Units were centrifuged at 1471×gfor 4 minutes and platelet-rich plasma and buffy coat were removed. Thepacked RBCs were diluted to an hematocrit of approximately 50% withErythrosol (ref 29), subsequently white cell reduced by using a filter(Leukotrap-SC RC, Pall Medical, East Hill, N.Y.), and oxygenated byadding 230 mL of a 60 to 40 percent O₂ to N₂ gas mixture to 150 mL of aRBC suspension in a 600 mL container (PL146 plastic, Baxter Healthcare)and by subsequent incubation for 30 minutes at 1 to 6° C. with agitation(orbital shaker, 100 r.p.m., 19-mm orbit, VWR Scientific, West Chester,Pa.). Oxygen levels were measured by use of a blood gas analyzer(Rapidlab 348, Bayer Corp., Medfield Mass.) and were routinelysupersaturated with levels greater than 400 mm Hg.

Addition of Virus and Phototreatment: Stock cultures of extracellularand intracellular viruses and Erythrosol were added to oxygenated, whitecell reduced, red cells to produce a final hematocrit of 20%. The volumeof the pathogen spike represented <10-percent of the total volume of thecell suspension. A volume of 10 mM aqueous stock of TMR-Se or 1 mMRhII-Se was added to 20% hematocrit red cells to yield final dyeconcentrations of 1 μM or 15 μM, respectively. For experiments conductedwith red cell suspensions containing dipyridamole, a volume of 10 mMaqueous stock of the quencher was added to virus containing red cellsprior to the addition of dye in order to yield a final concentration of200 μM. Red cell suspensions containing virus, dye, and, when indicated,dipyridamole were thoroughly mixed and divided into 2 mL aliquots inpolystyrene culture dishes (50 mm bottom diameter) to produce a 1 mmblood film. All treated and control samples contained dye but controlsamples were not illuminated. We incubated samples at room temperaturefor 15 minutes in the dark prior to illumination.

Because RBC storage studies required greater volumes than virus studies,21 petri dishes containing 2 mL each of 20-percent hematocrit RBCs withsensitizers and, when indicated, with dipyridamole, were illuminatedwith agitation on a reciprocal shaker (70 cycles/min, Melco EngineeringCorp., Glendale, Calif.), their contents pooled, and transferred to a150-mL PL145 container to provide sufficient volume for RBC storagestudies. RBC suspensions prepared at 20-percent hematocrit that did notcontain sensitizer or quencher and were not illuminated served ascontrols.

Samples were placed on an air-cooled transparent plastic stage andpositioned approximately 3 cm between two in-house fabricated lightbanks that illuminated samples from above and below. Each bank containedeight cool-white fluorescent bulbs (F20T12-CW, General Electric,Circleville, Ohio). For experiments using RhII-Se, light was filteredthrough amber 2422 plexigas plastic (with a cutoff circa 535 nm) toprevent absorption by dipyridamole, which may, in certain circumstances,behave as a photosensitizer (ref 30). The source produced broadbandlight at the sample surface of mW/cm² (without the amber filter) ormW/cm² (with the amber filter). Fluence rates were measured using ahandheld laser power meter with a silicon cell sensor (EdmundsIndustrial Optics, Barrington, N.J.).

Mammalian Virus Assays: VSV was provided by Med Lieu (HylandDiagnostics, Duarte, Calif.). Bovine virus diarrhea virus (BVDV) waspurchased from the American Type Culture Collection, Manassas, Va.).Pseudorabies virus (PRV) was provided by Shirley Mieka (American RedCross, Rockville, Md.).

Extracellular virus assays: VERO (isolated from African green monkeykidney, CCL81, ATCC) and MDBK (CRL6071, ATCC) cells were propagated inmedium (RPMI-1640 supplemented with glutamine, Biofluids, Rockville,Md.) supplemented with 10-percent bovine serum. Cells were seeded intosix-well culture plates and allowed to grow to confluency. Control andphototreated samples were serially diluted 10-fold, plated ontoconfluent VERO (for VSV and PRV) or MDBK (for BVDV) cell monolayers,incubated for 30 to 60 minutes, depending on the virus, and gentlyrocked at 37° C. for virus adsorption to cells. The inoculum was removedby aspiration and washed with PBS, a semi-liquid agar layer(0.2-percent) was added to each well and infected monolayers wereincubated at 37° C. in air containing 5-percent CO₂. Incubation periodswere: VSV, 1 day; PRV, 4 days; BVDV, 5 to 6 days. After incubation, theagar layer was removed by aspiration and the monolayer was stained with0.1-percent crystal violet in ethanol for at least 15 minutes. The stainwas removed by aspiration, the plates were washed with water, and theplaques enumerated.

Intracellular virus assays. Virus-infected cells were prepared byinoculating a confluent VERO monolayer with VSV at a virus-to—cell ratioof >1. The inoculum was incubated for 30 minutes at room temperature toallow entry of the virus and was subsequently removed by aspiration.Infected monolayers were washed with RPMI-1640 and detached from theculture flask by the addition of 0.05% trypsin and 0.02% versenesolution 5 minutes. The resulting infected cell suspension was dilutedapproximately 1 in 15 in RPMI-1640, and infected cells were centrifugedat 200×g for 10 minutes. The infected cell pellet was resuspended inadditive solution and subsequently added to white cell-reduced,oxygenated RBCs to give a final Hct of 45%. Control and phototreatedRBCs were first diluted 2-fold and then serially diluted 10-fold. The2-fold dilution was performed to eliminate RBC interference with VSVplaque formation. Infected cells were inoculated onto confluent,uninfected VERO cell monolayers in six-well plates. As in the assay forextracellular virus, semi-liquid agar (0.2%) was added to each well, andthe infected monolayers were incubated at 37° C. in air containing 5%CO₂ for 1 day. The agar layer was removed by aspiration, and themonolayer was washed with phosphate-buffered saline to remove RBCs andstained by the addition of 0.1% crystal violet in ethanol for at least15 minutes. After removal of the stain by aspiration, plates were washedwith water. Plaques were counted, and they represented viral growtharising from intracellularly infected VERO cells.

Red Cell Assays: Supernatant Hb was determined by thetetramethylbenzidine method. Total hemoglobin was determined by anautomated cell counter (Cell Dyn 3700, Abbott Laboratories, Abbott Park,Ill.). Extracellular potassium was measured using a blood gas andelectrolyte analyzer (RapidLab 348, Bayer Corp).

TMR-Se was observed to have extremely high extracellular virucidalactivity with little apparent hemolysis during storage. Only 1 μM ofcompound and 2 J cm⁻² of light were required to inactivate >7.4 log₁₀ ofextracellular VSV in 20% hematocrit red cells. With 45% hematocrit redcells, >7.4 log₁₀ of extracellular VSV was inactivated with 5 μM TMR-Seand 2 J cm⁻² of light. Under these conditions, there was littlehemolysis (0.22 and 0.36% respectively) during 42-day storage. Day 1potassium release was moderate (10 mM supernatant values) with samplestreated at 20 hematocrit and high (45% supernatant values) with samplestreated at 45% hematocrit. TMR-Se did not activate intracellular VSV(only 0.5 log₁₀ inactivation) under these conditions.

Similar to TMR-Se, TMR-S also could inactivate extracellular virus butrequired 5 times higher concentrations and more light exposure. Morethan 8 log₁₀ of extracellular VSV was inactivated using 5 μM TMR-S and7.4 J cm⁻² light in 20% hematocrit red cells. However, only 1.5 log₁₀ ofintracellular VSV could be inactivated under more stringent conditions(50 μM and 7.4 J cm⁻²); however, massive and immediate hemolysis (>>1%)was visually observed immediately after phototreatment under theseconditions.

With respect to RhII-Se—moderate levels of RhII-Se were required forextracellular and intracellular VSV inactivation. More than 8.5 log₁₀ ofextracellular and >6.5 log₁₀ of intracellular VSV were inactivated using15 μM RhII-Se and 2.5 J cm−2 light in 20% hematocrit red cells. Underthese conditions, there was little hemolysis (0.30%) during 42-daystorage. Potassium release in phototreated red cells was low, with 5.5mM potassium released to the supernatant after 1 day of storage.

The extremely high extracellular and intracellular inactivation levelsand low hemolysis during storage with RhII-Se is unexpected. In a studyof over 50 photosensitizers, it was observed that none could inactivatevirus to this degree without significant hemolysis during storage. Asubsequent study investigating the potential of RhII-Se to bind to thered cell membrane suggests that lack of binding to the red cell membranemay be the reason why so little hemolysis is observed followingphototreatment.

EXAMPLE 9

This example suggests that compounds which have an aryl group as asubstituent in the 9-position have higher virucidal activity than thosewith other groups, particularly methyl as substituent in the 9-position.In a comparative experiment, a compound 2 (FIG. 3) was synthesized,which differed from the other derivatives in that the 9-substituent isnot an aryl group (methyl in this example). This sample showed reducedvirucidal activity in either extracellular (<2.5 log₁₀) or intracellular(<0.5 log₁₀) testing over a wide range of concentrations.

The following examples describe the ability of the compounds of thepresent invention (such as TMR-O, TMR-S, and TMR-Se) to be transportedby P-glycoprotein (Pgp) into chemo-resistant CR1R12 cells. The resultsindicate that these compounds can be used as potent photosensitizers forPDT and may simultaneously act as reversal agents of Pgp function.

Experimental: Chemicals and reagents were purchased from Sigma AldrichChemical Co. (St. Louis, Mo.) unless otherwise noted. Cell culture mediaand antibiotics were obtained from Grand Island Biological (GrandIsland, N.Y.). Fetal bovine serum (FBS) was purchased from AtlantaBiologicals (Atlanta, Ga.). Tetramethylrosamine and Calcein AM (calceinacetoxymethyl ester) were purchased from Molecular Probes (Eugene,Oreg.). Both TMR-S and TMR-Se were prepared as described herein.

Cells and culture conditions: Cultured cells used in this study were theChinese hamster ovary parental cell line AUXB1, [47] a chemo-sensitivecell line in which Pgp content is very low and the multidrug resistantcell line CR1R12 which highly constituitively expresses Pgp. Multidrugresistance in CR1R12 cells were established from the CH^(R)C5 cell line[46] by sequential culturing in increasing concentrations of colchicinewith 5 μg mL⁻¹ being the final concentration used. Cell lines weremaintained in passage culture on 60-mm diameter polystyrene dishes(Corning Costar, Corning, N.Y.) in 4.0 mL Minimum Essential Medium(α-MEM) supplemented with 10% fetal bovine serum (FBS), 50 units mL⁻¹penicillin G, 50 ug ml⁻¹ streptomycin and 1.0 ug mL⁻¹ Fungizone®(complete medium). Only cells from passages 1-10 were used forexperiments. A stock of cells, passages 1-4, were maintained at −86° C.to initiate the experimental cultures. Cultures were maintained at 37°C. in a 5% CO₂ humidified atmosphere (Forma Scientific, Marietta, Ohio).Passage was accomplished by removing the culture medium, adding a 1.0 mLsolution containing 0.25% trypsin, incubating at 37° C. for 2 to 5minutes to remove cells from the surface followed by seeding new culturedishes with an appropriate number of cells in 4.0 mL of α-MEM. Cellcounts were performed using a particle counter (Model ZM, CoulterElectronics, Hialeah, Fla.).

EXAMPLE 10

Measurement of dye uptake into cell monolayers: Cell lines, AUXB1 orCR1R12, were seeded on 96-well plates in 200 μL/well α-MEM at 1-4×10⁴cells/well. Twenty four hours after seeding, verapamil at 7.0×10⁻⁶ M wasadded to selected cells in complete medium and cultures were incubatedin the dark at 37° C. for 2 h. Dyes were then added to the cultures at1×10⁻⁵ M in complete medium. Cells were incubated in the dark forselected times in the presence of each dye with or without verapamil forthe time course study and for 2 hr for comparative studies. The mediumwas then removed and the monolayers washed once with 200 μL 0.9% NaCland an additional 200 μL 0.9% NaCl was then added. The fluorescence ofthe intracellular dye was then determined using a multi-wellfluorescence plate reader (Gemini, Molecular Devices, Palo Alto,Calif.). The excitation/emission wavelengths were set at 490/570 forTMR-O and 540/600 for TMR-S. Because of the weak fluorescence signalemitted by TMR-Se, we were unable to obtain uptake data for thisanalogue. Intracellular dye concentration is expressed as fmole/cell.

EXAMPLE 11

Photoradiation of Cell Cultures: Cell lines, AUXB1 or CR1R12, wereseeded on 96-well plates in 200 μL/well α-MEM at 1-4×10⁴ cells/well.Rhodamine analogues were then added directly to the cell culture mediumat various concentrations and incubated for 2 h in the dark as above.The medium was then removed and 200 μL α-MEM minus FBS and phenol red(clear medium) were added to each well. One plate, with the lid removed,was then exposed to 350-750 nm light delivered at 1.4 mW cm⁻² for 1 h(5.0 J cm⁻²) from a filtered halogen source while a parallel plate waskept in the dark during the irradiation period. Immediately followingirradiation the clear medium was replaced with complete medium and themonolayers were incubated for an additional 24-h period. Subsequently,cells were trypsinized and counted using the Coulter counter todetermine cell viability. Determination of cell viability by cellcounting is performed according to an earlier method. [48] Briefly,cells that detach from the surface of a culture plate stain with trypanblue, i.e. are nonviable, while all cells that remain attached to thesurface exclude trypan blue, 100% viable. We were unable to seed newculture plates with cells that detached from the surface aftertreatment, while cells that remained attached after treatment continuedto grow and multiply. Thus, we count only those cells that remainattached to the culture plate surface after treatment and compare thosenumbers with the cell numbers obtained from control cells, cells notexposed to either dye or light. Data are then expressed as percent cellviability, treated cell counts/control cell counts.

EXAMPLE 12

Calcein AM uptake: CR1R12 or AUXB1 cells were seeded on 96-well platesin 200 μL/well α-MEM at 1-4×10⁴ cells/well. Rhodamine analogues at1.0×10⁻⁶ M were added to cell cultures on 96-well plates in completemedium for 2 h in the dark at 37° C. The medium containing the dyes wasremoved and replaced with clear MEM. A plate, with the lid removed, wasthen exposed to 350-750 nm light delivered at 1.4 mW cm⁻² for 1 h (5.0 Jcm⁻²) from a filtered halogen source. Another plate exposed to the sameculture and dye incubation conditions was kept in the dark. Also, inanother series of experiments, the Pgp modulators verapamil at 7.0×10⁻⁶M or 7.0×10⁻⁵ M or cyclosporin A at 7.0×10⁻⁶ M were incubated with thecells in the complete medium for 15 min. Immediately followingirradiation or incubation with the modulators, Calcein AM was added toeach well at 1.0×10⁻⁶ M in clear medium. The appearance of afluorescence signal at 530 nm, excitation 485 nm, was then followed inthe plate reader over a 30 min time period. The rate of uptake wascalculated from the slope of the line obtained in each experimentalprotocol and the data are expressed as percent of control Calcein AMuptake, the rate of Calcein AM taken up into cells that were not exposedto TMR analogues or Pgp modulators.

EXAMPLE 13

Time Course of Uptake of TMR-O into CR1R12 Cells: The development of thefluorescence signal from TMR-O at 570 nm was followed in cultured CR1R12cells either exposed to 7×10⁻⁶ M verapamil 2 h prior to dye addition orin cultures without previous exposure to verapamil. The data in FIG. 6clearly show that verapamil enhances the uptake of TMR-O into CR1R12cells compared to those cultures not previously exposed to verapamil.The increase in intracellular uptake of TMR-O in the presence ofverapamil becomes statistically significant (P<0.005) by 1 h after dyeaddition to monolayers of cells.

EXAMPLE 14

Comparison of TMR-O and TMR-S uptake into A UXB-1 or CR1R12 cells: TMR-Oor TMR-S was added for 1 h at 1×10⁻⁵ M to cultured AUXB-1 or CR1R12cells, either alone or after cells had been incubated with 7×10⁻⁶ Mverapamil for 2 h as above. The data in FIG. 7 demonstrate that theintracellular accumulation of either TMR-O or TMR-S was similar in AUXB1cells in the absence or the presence of verapamil. In CR1R12 cells, theaccumulation of both TMR-O and TMR-S was significantly enhanced in thepresence of verapamil compared to cells not previously exposed toverapamil. A more than 3-fold increase in uptake for TMR-S was obtained.The uptake of TMR-S in the chemo-resistant cell line, CR1R12, in thepresence of verapamil was equivalent to its uptake in thechemo-sensitive parent cell line AUXB1 in the absence or presence ofverapamil. In AUXB1 cells in the absence or presence of verapamil and inCR1R12 cells in the presence of verapamil, the uptake of TMR-O wasequivalent to the uptake of TMR-S. These data clearly show thatverapamil, a strong modulator of Pgp function, significantly effects theuptake of TMR-O or TMR-S in the chemo-resistant cell line CR1R12 whileno effect was seen in the chemo-sensitive AUXB1 cell line. Under theconditions employed, the results strongly suggest that TMR-S is atransport substrate for Pgp, similar to TMR-O.

EXAMPLE 15

Phototoxicity toward CR1R12 chemo-resistant cells: The data in FIG. 8depict the results obtained when CR1R12 cells are exposed to TMRanalogues and 5.0 J cm⁻² light with or without 7×10⁻⁶ verapamil. Thedata clearly demonstrate that none of the TMR analogues are effective asphotosensitizers of CR1R12 cells in the absence of previous exposure toverapamil. However, when TMR-S and TMR-Se were added to CR1R12 cellsafter verapamil exposure for 2 h, irradiation of cultures with broadband white light caused significant cytotoxicity. TMR-O showed nosignificant phototoxicity in the absence or presence of verapamil evenat the highest concentration, presumably due to its relatively lowsinglet oxygen production. [19] These data show that TMR-S and TMR-Seare phototoxic to chemo-resistant CR1R12 cells when a Pgp modulator ispresent indicating that uptake ensues and that the dyes are able toreach and cause damage to a critical intracellular sites duringirradiation.

EXAMPLE 16

Comparison of TMR-Se phototoxicity towards AUXB1 or CR1R12 cells: Forcomparison studies, parallel phototoxicity experiments were performedboth with AUXB 1 cells, which have low levels of Pgp, and with CR1R12cells, which constituitively express high levels of Pgp. The datadisplayed in FIG. 9 demonstrate that the chemo-sensitive AUXB 1 cellsare equally susceptible to phototoxicity using TMR-Se with or withoutprevious exposure to 7×10⁻⁶ M verapamil while significant phototoxicitytowards CR1R12 cells only occurs when verapamil is present.

EXAMPLE 17

Effect of TMR-S or Se photosensitization on the intracellular uptake ofCalcein AM: Calcein AM (CAM) is a nonfluorescent, hydrophobic compoundthat readily crosses the plasma membrane of normal cells. Once insidethe cell, the ester bonds of CAM are enzymatically cleaved transformingit into the highly fluorescent, hydrophilic calcein. Appearance of thecalcein fluorescence signal following exposure of cells to CAM indicatesthe intracellular uptake of CAM followed by ester hydrolysis to formcalcein and retention of calcein in the cytosol of the cell.

Cells were exposed to TMR-S or TMR-Se (1×10⁻⁵ M) with or without lightexposure or to the Pgp modulators verapamil or cyclosporin A (CsA) inthe dark. Following exposure to the dyes with or without light or to themodulators, CAM was added at 1×10⁻⁶ M and its intracellular accumulationover 30 min was monitored by measuring the increase in calceinfluorescence at 530 nm with excitation at 485 nm. The data displayed inFIG. 10 show that the Pgp modulators verapamil and CsA increased theuptake of CAM into CR1R12 cells from 6 to 13 fold over control CAMuptake in the absence of any modulators. Exposure of CR1R12 cells toTMR-S or TMR-Se for 2 h in the dark resulted in no significant change inthe intracellular accumulation of CAM (data not shown). However, 1 h oflight exposure after incubation of cells with TMR-S or TMR-Se resultedin an up to 2-fold increase in CAM uptake.

Parallel experiments were performed with the parent cell line, AUXB1. Inthese chemo-sensitive cells, CAM in the presence of Pgp modulators wasaccumulated intracellularly at a rate equivalent to that obtained forcontrol AUXB1 cells, 1000±30 relative fluorescence units (RFU) ofcalcein/min/1×10⁵ cells. This rate of CAM uptake into AUXB1 cells was10-fold greater than that measured in control CR1R12 cells at 94±3RFU/min/1×10⁵ cells. Curiously, higher concentrations of TMR-S or TMR-Seplus light exposure virtually abolished CAM uptake into AUXB1 cells.This result could be due to factors such as damage to the plasmamembrane system(s) responsible for intracellular accumulation of CAM orinhibition of intracellular esterases responsible for cleaving the esterbond on CAM that provides a fluorescent compound. These data, takentogether, demonstrate that TMR-S and TMR-Se and light, under theconditions employed, do increase the intracellular accumulation of thePgp substrate CAM into chemo-resistant CR1R12 cells. The experimentalconditions remain to be optimized but these initial experimentsdemonstrate that highly expressing Pgp cells can be altered by thesetetramethylrosamine analogues to take in 2 times more Pgp substrate thancells not exposed to the dyes and light.

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1. A compound having the structure of Formula I:

wherein E is S and A⁻is an anionic group selected from the groupconsisting of halides, sulfonates; carboxylates; hexafluorophosphate,and tetrafluoroborate; and I) i) W, X, Y, and Z are hydrogen; ii) R₁′,R₂′, R₁″ and R₂″ are independently hydrogen or C₁ through C₈ branched orunbranched alkyl, and, optionally, 1) R₁′ and R₂′ are alkyl groupsconnected such that they comprise a 3-, 4-, 5-, 6- or 7-membered ring

 which, optionally, bears alkyl or aryl substituents; and/or 2) R₁″ andR₂″ are alkyl groups connected such that they comprise a 3-, 4-, 5-, 6-or 7-membered ring

 which, optionally, bears alkyl or aryl substituents; iii) R is an arylgroup which is mono-, di-, or tri-substituted with one or more of thefollowing substituents: —CO₂Me, —CO₂H, dialkylamino groups having alkylgroups which are independently C₁ through C₈; C₁ through C₈ alkyl, C₁through C₈ alkoxy, and heterocyclic groups; heteroaryl or substitutedheteroaryl group;

wherein R₄ and R₅ are C₁ through C₈ alkyl groups; wherein R is not aphenyl group mono-substituted in the ortho position or di-substituted inthe ortho and para positions bearing groups of the following form: —CO₂Hor CO₂R₄ or II) i) W, X, Y and Z are independently hydrogen or C₁through C₈ alkyl; ii) R₁′, R₂′, R₁″ and R₂″ are independently hydrogenor C₁ through C₈ alkyl, and wherein R₁′ and Y are connected such thatthey comprise a five, six or seven-membered ring

 or and/or R₂′ and Z are connected such that they comprise a five-, six-or seven-membered ring

 and/or R₁″ and W are connected such that they comprise a five-, six- orseven-membered ring

 and/or R₂″ and X are connected such that they comprise a five-, six- orseven-membered ring:

and iii) R is an aryl group which is mono-, di-, or tri-substituted withone or more of the following substituents: —CO₂Me, —CO₂H, dialkylaminogroups having alkyl groups which are independently C₁ through C₈; C₁through C₈ alkyl, C₁ through C₈ alkoxy, and heterocyclic groups,heterocyclic or substituted heterocyclic group.
 2. A compound as claim 1wherein R is an aryl group which is mono-, di-, or tri-substituted withone or more substituents selected from the group consisting of —NMe₂,—NHEt, —NH₂, -Me, —OMe, 1,3-oxazole, 1,3-diazole,4,5-dihydro-1,3-oxazole, and 4,5-dihydro-1,3-thiazole.
 3. A compound asin claim 1 wherein A⁻is an anionic group selected from the groupconsisting of Cl, tosylate; and acetate.
 4. A compound as in claim 1wherein R₁′, R₂′, R₁″, and R₂″ are methyl.
 5. A compound as claim 1wherein R₁′, R₂′, R₁″, or R₂″ is an ethyl, dimethylamino or ethylaminogroup.
 6. A compound as in claim 4, wherein R is an aryl group whichbears a heterocyclic substituent at position
 2. 7. A compound of claim 1wherein R₁′, R₂′, R₁″, and R₂″ are methyl, and R is2,4,6-trimethylphenyl.
 8. A compound of claim 7 wherein A⁻is chloride orhexafluorophosphate.